The present invention relates generally to the use of electric pulses to increase the permeability of cells, and more specifically to a method and apparatus for the application of controlled electric fields for in vivo delivery of pharmaceutical compounds and genes into cells by electroporation therapy (EPT), also known as cell poration therapy (CPT) and electrochemotherapy (ECT).
The skin is an especially attractive target for gene therapy. In particular, the ability to target genes to the epidermis of the skin could be used to correct skin-specific disorders as well as for the production of proteins secreted into the skin to correct certain systemic diseases. For example, genes expressing cytokines, interferons or other biologically active molecules could be used to treat skin tumors or other lesions. In addition, keratinocytes and fibroblasts in the skin can secrete protein factors which circulate to treat systemic conditions such as hemophilia. Despite the clear potential in using skin as a target for gene therapy, the major technical problem of an in vivo method of gene delivery remains largely unresolved. Since the stratum corneum (SC) acts as a significant physical barrier to gene transfer into the skin, the technical problem of how to deliver genes through this layer persists.
Similarly, muscle cells are also useful targets for gene therapy due to their ubiquity. Nonetheless, as with skin, there exists a need for a method to reliably introduce exogenous therapeutic material into muscle cells.
Gene therapy does not include only intrinsically therapeutic genetic material (i.e., genes that encode a missing or underexpressed gene product), but also those which elicit an immune response. One of the oldest and most effective forms of preventative care against infectious diseases is vaccination. Safe and effective vaccines are available to protect against a variety of bacterial and viral diseases. These vaccines consist of inactivated pathogens, recombinant of natural subunits, and live attenuated or live recombinant microorganisms.
DNA immunization, a novel method to induce protective immune responses, was recently introduced into the scientific community and proven to be very effective in animal models. This technology is currently in first safety and efficacy trials in human volunteers. DNA immunization entails the direct, in vivo administration of plasmid-based DNA vectors that encode the production of defined microbial antigens. The de novo production of these antigens in the host""s own cells results in the elicitation of antibody (i.e., humoral) and cellular immune responses that provide protection against live virus challenge and persist for extended periods in the absence of further immunizations. The unique advantage of this technology is its ability to mimic the effects of live attenuated vaccines without the safety and stability concerns associated with the parenteral administration of live infectious agents. Because of these advantages, considerable research efforts have focused on refining in vivo delivery systems for naked DNA that result in maximal antigen production and resultant immune responses.
The most widely used administration of vaccine DNA is direct injection of the DNA into muscle or skin by needle and syringe. This method is effective in inducing immune responses in small animals, as mice, but even here it requires the administration of relatively large amounts of DNA, ca. 50 to 100 ug per mouse. To obtain immune responses in larger animals, as rabbits, non-human primates, and humans, very large amounts of DNA have to be injected. It has to be seen whether this requirement for very large amounts of vaccine DNA turns out to be practical, for safety and commercial reasons, in human applications.
A cell has a natural resistance to the passage of molecules through its membranes into the cell cytoplasm. Scientists in the 1970""s first discovered xe2x80x9celectroporationxe2x80x9d, where electrical fields are used to create pores in cells without causing permanent damage to the cells. Electroporation made possible the insertion of large molecules directly into cell cytoplasm by temporarily creating pores in the cells through which the molecules pass into the cell.
Electroporation techniques (both in vitro and in vivo) function by applying a brief high voltage pulse to electrodes positioned around the treatment region. The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the implant agent enter the cells.
Electroporation has been used to implant materials into many different types of cells. Such cells, for example, include eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and sperm. Further-more, electroporation has been used to implant a variety of different materials, including nucleic acids, polypeptides, and various chemical agents.
Electroporation ha s been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the implant agent and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture.
With in vivo applications of electroporation, electrodes are provided in various configurations such as, for example, a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, needle-shaped electrodes may be inserted into the patient, to access more deeply located cells. In either case, after the therapeutic agent is injected into the treatment region, the electrode s apply an electrical field to the region. In some electroporation applications, this electric field comprises a single square wave pulse on the order of 100 to 500 V/cm, of about 10 to 60 ms duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820, made by the BTX Division of Genetronics, Inc.
Electroporation has been recently suggested as an alternate approach to the treatment of certain diseases such as cancer by introducing a chemotherapy drug directly into the cell. For example, in the treatment of certain types of cancer with chemotherapy it is necessary to use a high enough dose of a drug to kill the cancer cells without killing an unacceptable high number of normal cells. If the chemotherapy drug could be inserted directly inside the cancer cells, this objective could be achieved. Some of the best anti-cancer drugs, for example, bleomycin, normally cannot penetrate the membranes of certain cancer cells effectively. However, electroporation makes it possible to insert th e bleomycin into the cells.
Despite the suitability of the epidermis as a target tissue for gene therapy, there are significant barriers to safe, easy, efficient, and economical gene delivery. In particular, the lipid-rich SC, which is composed of dead keratinocytes surrounded by multiple, parallel bilayer membranes, represents a formidable physical barrier to epidermal gene transfer. To overcome this barrier a novel, non-viral approach, involving the basic concept of electroporation to introduce genes into the epidermis is provided by the present invention.
As described above, the technique of electroporation is now a well-established physical method for transfection of cells that allows introduction of marker molecules, drugs, genes, antisense oligonucleotides and proteins intracellularly. However, there still exists a need to introduce therapeutic agents directly into skin cells or through the skin and into muscle cells without direct injection.
The present invention describes an in vivo method, using pulsed electric field to deliver therapeutic agents into cells of the skin and muscle for local and systemic treatments. In particular, therapeutic agents include naked or formulated nucleic acid, polypeptides and chemotherapeutic agents.
Therapeutic agents can be employed directly as palliative agents (i.e., those which directly exert a therapeutic effect), or as agents with a less direct effect (e.g., genes encoding polypeptides that elicit an immune response).
The advantages offered by electroporation for skin and muscle-directed gene therapy include: (1) elimination of the risk of generating novel disease-causing agents, (2) delivery of DNA molecules much larger than can be packaged into a virus, (3) no immune responses or toxic side effects by non-DNA material, e.g., viral proteins or cationic lipids, (4) DNA enters the cell through a non-endosomal pathway, diminishing the rate of DNA degradation, and (5) the method is simple, highly reproducible and cost-effective.
In accordance with one embodiment of the present invention, there is provided an in vivo method for introducing a therapeutic agent into skin cells of a subject, comprising applying a pulsed electric field to the skin cells substantially contemporaneously with the application of therapeutic agent to the skin cells, such that the therapeutic agent is introduced into the skin cells.
In accordance with another embodiment of the present invention, there is provided a method for inducing an immune response in a subject, comprising applying a pulsed electric field to skin and/or muscle cells of the subject substantially contemporaneously with the application of an immune response-inducing agent to the skin and/or muscle cells, such that the immune response-inducing agent is introduced into the skin and/or muscle cells thereby inducing in the subject an immune response.
In accordance with still another embodiment of the present invention, there is provided a method for the therapeutic application of electroporation to skin and/or muscle cells of a subject for introducing topically applied molecules into said cells, comprising providing an array of electrodes, at least one of the electrodes having a needle configuration for penetrating tissue; inserting the needle electrode into selected tissue; positioning a second electrode of the array of electrodes in conductive relation to the selected tissue; and applying pulses of high amplitude electric signals to the electrodes, proportionate to the distance between the electrodes, for electroporation of said tissue; such that said topically applied molecules are introduced into said skin and/or muscle cells.
In another embodiment of the present invention, there is provided a micropatch electrode for use with an electroporation apparatus, said micropatch electrode having a substantially planar array of patch elements, each patch element comprising two sets of electrodes, wherein each set of electrodes comprises a first electrode and a second electrode electrically insulated from one another, such that when different electric potentials are applied to said first and second electrodes, a voltage is produced therebetween.
An electrode kit for use in conjunction with electroporation therapy, said kit having a micropatch electrode as described herein, and an injection needle, optionally comprising one or more holes disposed along its length and proximal to the needle tip, wherein said holes are in fluid communication with the hollow interior of said injection needle.
In accordance with another embodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having a ring-shaped electrode having an electrically insulating shield, wherein the electrically insulating shield provides support to the ring-shaped electrode and electrically insulates a tissue under treatment employing the electrode, and an electrically conducting injection needle, optionally comprising one or more holes disposed along its length and proximal to the needle tip, wherein the holes are in fluid communication with the hollow interior of the injection needle, wherein a potential difference applied to the ring-shaped electrode and the needle electrode creates a voltage therebetween.
In accordance with yet another embodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having an array of a plurality of paired electrode needles and, at leas t one injection needle, wherein a potential difference applied said paired electrode needles creates a voltage therebetween.
In accordance with another embodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having a suction generating device comprising a ring electrode disposed about an injection needle electrode, such that when said ring electrode is contacted with skin of a subject a and suction is generated by the suction generating device, the skin is pulled up around the injection needle electrode, causing the injection needle electrode to pierce the skin, and wherein a potential difference applied to the ring-shaped electrode and said injection needle electrode creates a voltage therebetween.
In accordance with another embodiment of the present invention there is provided an electrode for use with an electroporation apparatus, said electrode having an injection needle and paired electrically conducting wires disposed within the hollow core of the injection needle and protruding from the tip thereof, the electrically c conducting wires having an electrically insulated portion and an exposed electrically conducting portion, the exposed electrically conducting portion being at the end of said wires protruding from the tip of said injection needle.
In accordance with another embodiment of the present invention there is provided a needle electrode for use with an electroporation apparatus, said needle electrode having disposed around a portion of its length a substance-releasing material.
In accordance with another embodiment of the present invention there is provided a needle electrode for use with an electroporation apparatus, said needle electrode having a hollow central core, at least four holes along its length, wherein the four holes comprise two paired holes, each of said two paired holes comprising a hole proximal to the tip of the needle and a hole distal to the tip of said needle, and a pair of electrically conducting wires having an electrically insulated portion and an exposed electrically conducting portion, wherein the pair of electrically conducting wires are located in said hollow core, except for the exposed electrically conducting portion which runs outside of the needle, extending outward from the distal paired hole to the proximal paired hole where it re-enters the hollow core.
In accordance with another embodiment of the present invention there is provided a needle electrode for use with an electroporation apparatus, said electrode having a plurality of electrically conducting needles disposed within a depth guide, wherein the tip of the needles extend for a predetermined distance beyond the depth guide.